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S Harayama

Publications and source records attributed to S Harayama.

At least 127 records · Page 7Linked to original sources
PubMed1984

Structure of the Trg protein: Homologies with and differences from other sensory transducers of Escherichia coli.

Transducer proteins are central to chemotaxis in Escherichia coli. Three transducer genes comprise a homologous gene family, while a fourth gene, trg, is more distantly related. We have determined the nucleotide sequence of trg. The deduced sequence of the Trg protein has features in common with other transducers as well as regions of significant divergence. The protein sequence suggests the same transmembrane structure postulated for other transducers: an extra cytoplasmic NH2-terminal domain connected by a membrane-spanning region to an intracellular COOH-terminal domain. The COOH-terminal domain of Trg exhibits substantial sequence identity with the corresponding domains of the other transducers, particularly near the sites of covalent modification. Trg appears to have the same five methyl-accepting sites identified in the Tsr protein. Two of those sites are glutamines that are deamidated to yield methyl-accepting glutamates, while the remainder are synthesized as glutamates. Conservation in number but not in position of modified glutamines in Trg compared to the other transducers is consistent with the notion that uncharged glutamines at a specific number of modification sites serve to balance the signaling state of newly synthesized transducers. The NH2-terminal domain of Trg exhibits no significant homology with other transducers, implying that trg may be a fusion of the common COOH-terminal transducer sequence with an unrelated NH2-terminal sequence. The location of specific mutations within trg provides support for the suggestion that ligand-binding sites are in the NH2-terminal domains.

Amino Acid Sequence↗
PubMed1984

Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12.

We isolated spontaneous and transposon insertion mutants of Escherichia coli K-12 that were specifically defective in utilization or in high-affinity transport of D-ribose (or in both). Cotransduction studies located all of the mutations near ilv, at the same position as previously identified mutations causing defects in ribokinase ( rbsK ) or ribose transport ( rbsP ). Plasmids that complemented the rbs mutations were isolated from the collection of ColE1 hybrid plasmids constructed by Clarke and Carbon. Analysis of those plasmids as well as of fragments cloned into pBR322 and pACYC184 allowed definition of the rbs region. Products of rbs genes were identified by examination of the proteins produced in minicells containing various rbs plasmids. We identified four rbs genes: rbsB , which codes for the 29-kilodalton ribose-binding protein; rbsK , which codes for the 34-kilodalton ribokinase ; rbsA , which codes for a 50-kilodalton protein required for high-affinity transport; and rbsC , which codes for a 27-kilodalton protein likely to be a transport system component. Our studies showed that these genes are transcribed from a common promoter in the order rbsA rbsC rbsB rbsK . It appears that the high-affinity transport system for ribose consists of the three components, ribose-binding protein, the 50-kilodalton RbsA protein, and the 27-kilodalton RbsC protein, although a fourth, unidentified component could exist. Mutants defective in this transport system, but normal for ribokinase , are able to grow normally on high concentrations of the sugar, indicating that there is at least a second, low-affinity transport system for ribose in E. coli K-12.

Bacterial Proteins↗
PubMed1984

Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.

DNA Restriction Enzymes↗
PubMed1983

Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning.

We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein. The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC. The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form. The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane. Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon. The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations. The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose. The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.

Bacterial Proteins↗
PubMed1982

A gene coding for a periplasmic protein is located near the locus for termination of chromosome replication in Escherichia coli.

Hybrid plasmids carrying trg, the genetic locus in closest proximity to terC, coded for several polypeptides in addition to the Trg protein. Polypeptides of 59,000 and 61,000 apparent molecular weight were the most prominent products synthesized in minicells containing the hybrid plasmids. Analysis of the effects of deletions generated by a restriction endonuclease identified a region of DNA immediately adjacent to trg as the putative gene coding for the two polypeptides. Studies with whole cells and minicells showed that the 59,000-dalton polypeptide is a periplasmic protein. Analysis by limited proteolysis indicated that the two polypeptides are related, and a number of observations support the notion that the 61,000-dalton protein is a precursor form of the 59,000-dalton mature exported protein. The identification and characterization of a protein, in addition to Trg, which is produced by a gene in close proximity to terC emphasizes the fact that the region does contain intact and active genes.

Bacterial Proteins↗
PubMed1982

Cloning of trg, a gene for a sensory transducer in Escherichia coli.

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.

Bacterial Proteins↗
PubMed1982

Studies on the export of the maltose-binding protein and the LamB protein.

Two proteins encoded in the malB locus, the maltose-binding protein and the LamB protein, are exported to the periplasm and outer membrane, respectively. Both proteins are synthesized on membrane-bound polysomes in precursor forms which contain signal sequences at their amino-termini. Proteolytic removal of the signal sequence from maltose-binding protein begins co-translationally but is nat initiated until the nascent chains have attained 80% of their final length. Approximately one-third of the molecules are processed co-translationally, the remainder being processed post-translationally. In contrast, the LamB precursor is processed almost entirely post-translationally. In neither case is it clear when the polypeptide chains are transferred through the cytoplasmic membrane. An interesting feature with as yet unknown significance is the non-uniform rate of elongation of maltose-binding protein. The export of maltose-binding protein and the lamB protein as well as that of other proteins, has been shown to require an energized membrane.

ATP-Binding Cassette Transporters↗
PubMed1981

Energy is required for maturation of exported proteins in Escherichia coli.

It has been established in numerous cases that proteins which are exported from Escherichia coli are synthesized on membrane-bound polysomes in precursor forms which are proteolytically cleaved to generate the mature species. Here we present evidence that at least one step in the export of proteins requires energy. Energy requirements for processing of the precursors of both the M13 coat protein [Date, T., Zwizinski, C., Ludmerer, S., and Wickner, W. (1980) Proc. Natl Acad. Sci. USA, 77, 827-831; Date, T., Goodman, J. M., and Wickner, W. T. (1980) Proc. Natl Acad. Sci. USA, 77, 4669-4673] and the B subunit of heat-labile enterotoxin [Palva, T., Hirst, T. R., Hardy, S. J. S., Holmgren, J., and Randall, L. L. (1981) J. Bacteriol. in the press] have been demonstrated previously. An energy requirement for the proteolytic processing of an additional five exported proteins is reported here. Studies utilizing an uncA mutant suggest that the form of energy required is proton-motive force. Thus an energized membrane is probably essential for export of most periplasmic and outer membrane proteins.

Adenosine Triphosphate↗
PubMed1981

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles.

We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage lambda receptor protein in its outer membrane. Phage lambda adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using lambda vehicles carrying transposons. This system can also be used for cosmid cloning.

Bacterial Outer Membrane Proteins↗
PubMed1981

Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4.

A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30 degrees C but not at 42 degrees C, clones are isolated resistant to kanamycin at 42 degrees C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30 degrees C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42 degrees C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained. About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10(-10). The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn1 in many Gram-negative bacterial species.

DNA Transposable Elements↗
PubMed1981

Methyl-accepting chemotaxis protein III and transducer gene trg.

A comparison of the two-dimensional gel patterns of methyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiple methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed.

Bacterial Proteins↗
PubMed1980

High frequency mobilization of the chromosome of Escherichia coli by a mutant of plasmid RP4 temperature-sensitive for maintenance.

Two mutants of plasmid RP4 temperature-sensitive for maintenance were isolated and one of them (pTH 10) was extensively studied. Cells carrying pTH10 showed temperature-sensitive drug resistance from which we isolated a number of temperature-independent derivatives. Almost all of them were Hfrs donating chromosomal genes to recipient bidirectionally from different points of origin. The Hfrs may be formed in two steps: (1) the transposon (Tn 1) carried by pTH 10 translocates into the host chromosome, and (2) pTH 10 is integrated in the host chromosome by reciprocal recombination between the TN 1 s, one situated on pTH 10 and another on the host chromosome. That temperature-independent drug resistance selects for this type of derivative, was supported by the following observations: (1) Hfrs thus obtained were usually unstable and segregated at high frequency 'revertants' showing temperature-sensitive drug resistance when they were cultivated at 30 degrees C. (2) The 'revertants' cured of pTH 10 were still ampicillin resistant, indicating existence of Tn 1 inserted in the host chromosome. (3) Tn 1 insertions found in these derivatives mapped in the vicinity of points of origin of the original Hfrs. (4) When new Hfrs were constructed by: (a) transduction with Plkc on Tn 1 insertions found in derivatives of Hfrs, (b) introduction of pTH 10 into the transductants,and (c) isolation of clones of temperature-independent drug resistance from such pTH 10 carrying stains, they had similar characteristics to the original Hfrs from which Tn 1 insertions were derived. Possibilities for genetic manupulation using pTH 10 in a wide range of Gram-negative bacteria are discussed.

Chromosomes, Bacterial↗
PubMed1979

Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man. We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable. The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.

Chemotaxis↗
PubMed1979

Mutants in transmission of chemotactic signals from two independent receptors of E. coli.

We have characterized chemotactic mutants of E. coli that appear to be defective in a common linkage of two independent receptors to the central chemotactic components. The mutants do not respond to gradients of ribose or galactose and thus are called trg (taxis to ribose and galactose), after Ordal and Adler (1974b). These trg mutants are indistinguishable from their parent in tactic response to other attractants, swimming pattern, growth rates, and transport of ribose and galactose. The mutant cells contain the usual amounts of ribose and galactose receptors, and those proteins function normally in their other role, transport of their respective ligands. The mutations, generated by insertion of translocatable drug-resistance elements (transposons)8 are located near 31 min on the map of the E. coli chromosome, a locus far removed from the genes coding for the ribose and galactose receptors. Trg mutants do not resemble either specific receptor mutants or che mutants. The nature of the requirement for the trg product in the response to ribose and galactose is not defined, but evidence for interference of tactic signals from the ribose and galactose receptors (Strange and Koshland, 1976) supports the idea that the product functions directly in the transmission of tactic signals from the two receptors to the flagella.

Biological Transport↗
PubMed1979

Relation of chemotactic response to the amount of receptor: evidence for different efficiencies of signal transduction.

We determined the content of galactose-glucose-, maltose-, and ribose-binding proteins in cells of Escherichia coli K-12 grown in a variety of media and also measured the respective transport and chemotactic activities that depend on those binding proteins. Correlation of the level of induction of a particular binding protein with the extent of tactic activity mediated by that protein indicates that the magnitude of the tactic response to a particular stimulating compound is a direct function of the number of receptors per cell. In contrast, comparison of the magnitudes of response to substances recognized by independent receptors indicates that some stimulus-receptor complexes are more effective in eliciting tactic responses than are others. Thus, the magnitude of response to any particular stimulating compound is a function both of the number of receptors per cell and of the effectiveness of the stimulus-receptor complex. Considerations of available information about the tactic response to maltose suggest that the effectiveness of a stimulus-receptor complex is related to the transducer with which the receptor interacts. The tar product appears to be a relatively effective transducer of the signals it accepts from receptors for aspartate, alpha-methylaspartate, and maltose, whereas the trg product appears to be a relatively ineffective transducer of signals it accepts from receptors for galactose and ribose.

Bacterial Proteins↗
PubMed1977

Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off" light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.

Aerobiosis↗